Antibiotic pactacin and method of production



May 11, 1965 A. D. ARGOUDELIS ETAL ANTIBIOTIC PACTACIN AND METHOb 0FPRODUCTION Filed Oct. 2, 1961 m ar' FIGWE l. WRARED ABSORFTDN SPECTRW-PACTACII IN Old" I WAVE CLARENCE DE BOER THOMAS E. EBLE ROSS R. HERRALEXANDER D. ARGOUDEU INVENTOR.

Afro/Web United States Patent "ice Patented M a 3 TABLE Ill-CULTURALCHARACTERISTICS 3,183,154 ANTIBIOTIC PACTACIN AND METHOD OF PRODUCTION Madium Surface Reverse Other Alexander D. Argoudells, Clarence De Boer,Thomas E.

Eble, and Ross R. Herr, all of Kalamazoo, Mich., 5 assignors to TheUpjohn Company, Kalamazoo, M mam gelatin Pam gray a corporation ofDelaware faction.

Filed Oct. 2, 1961, Ser. No. 142,192 331; 533 Gray-WNW gg z- W et l a 11Claims. (Cl. 167-65) mom five i droppingtohase. This invention relatesto a novel composition of matter pigment ore nation. a to a P Prroductwn thereof Mare P Smhmmm, mega? Smmpemm ticularly, this inventionrelates to a new compound, pacbmni. white. Slight growth tacin, and to aprocess for the production thereof. Pactacin is a biosynthetic productobtained as an elabgigrpent, ncnon. oration product of a pacta m-pro mga m my 15 mmusmnk "do Blue surface pemm It is an amphoteric substancewhich has a property of P e pgo nzation;

p advfirsdy P15801913 the growth of ccrtfim par Peptonelron agar. Palegray-pink- Yellow-tan.-. N0 H38 darkening. ticularly bacteria, forexample, BdCllIllS subtzlis, Pseu- Calcium malate rale g r Palehgray-Mfllillttgliotd agar. w i w ite. sou iize domorras aerugmosa, Klebslellapneumomae, Proteus skim milk gum Pale Yellow yellow pigment vulgarls,and Salmonella typhl and can be used along or {Design hydrowe incombination with other antibacterial agents to prevent Gh-mse spamPaleluvendeb Pale new PM? yellow, the growth of or reduce the number ofsuch organisms gmeagar. Priink. film. p igme it. present in variousenvironments. For example, it is useymme z gg eye f g g 'l' ful tocontrol the infection of silk worms caused by sl'rgsiined pathogeniccultures of B. subtilis; it is also useful as an Xanthlne man" Creamfl,.,,,,;- oil preservative, for example, as a bacteriostatlc agentgllgrggnt. th to inhibit the growth of certain microorganisms that causesoiiiblli iioli spoilage in cutting oils. under growth. The actinomyceteused according to this invention Mmose tlf g fgfigig. pigment for theproduction of pactacin has been designated as Ben at m w lt tufts. d Y nt I Slrepiomyces pactum var. pactum. One of its strain charn as T 2 2;:he acteristics is the production of pactacin. A subculture of whiteaerial at l8 and 24"; blue this variety can be obtained from thepermanent collecmy 28am tion of the Northern Utilization and ResearchDivision, 17236532201; d Agricultural Research Service, US. Departmentof Agri- 24 i culture, Peoria, Illinois, USA. Its accession number ingP- this repository is NRRL 2939. F 8

um 1 Czapeks sucrose Pale gray- Pale grey- No pigment. Pale Streptomycespactum var pact has gray to b ulsh agar. white white. flayqhm mm grayaerial growth and gray to grayish olive reverse on M18, 24. as"; mostmedia. Spores are borne in very compact spirals 40 rr v t 3 xellowreverse and are covered with fine hairs. Its growth characteristics at13 and 2 1 on standard biological media and its carbon assimilation ggeg g are given in the following tables. at 5 N0 growth at TABLEI.APPEARANCE OF EKTACHROMIE, 45

t S Lee R l medmm q The new compound of the invention is produced when 1i any We Tan the elaborating organism is grown in an aqueous nutrient 2:TruCt gn filiirif'ii (wildnes medium under submerged aerobic conditions,and preferif ggff f gg g;- ably in a nutrient medium containing a carbonsource, 51 o. i% t n-s-m6 T XlK'G graywhito Pale yellow. for example, anassimilable carbohydrate, and a nitromsemsmrcn gen source, for example,an assimilable nitrogen com- H pound or proteinaceous material. It isto'be understood 1 l)1etz, A Ektlchrmnc lmnspnrent-ies as :nds lllactnlomycote ClLlSSlfieation, Annals oi the N.Y. Academy oiScienoe152454, 1954. 55 3150 that for thC prepa a ion o l m lcd amountsSllIfElCfi TABLE Il.ASSlMlLATlON OF CARBON COM POUNDS lN SYNTHETICMEDIUM D-xyloso (+l l (clln lose Salicin L-arabinose ltnlllnose PhenolRhmnnose (4-) Dcxtrin (.resol D-iructnse 5 lnulin Na tormate D-gnlaotosel Soluble starch Na oxalate t+) ll-glucose l llyccrnl N a tartratcD-lnannose Dnlcitol (l+)(+) Ilia snlicylate Mnltose K l)-innnnito aacetate Sucrose (-H ll-sorhitol No citrate mtrogenous Sources Lactose iInositol Na succinate Control lrldhnm. T. G.. and Gottlleb. I1,Assimilation of Carbon Compounds in Synthetic Medium, J. Bact. 56:107-114, 1948.

+ Positive assimilati n.

(-l-l Positive nssimilatiou-only slight growth.

() Slight. growth-no assimilation.

cultures in bottles can be employed. Preferred carbon sources includeglucose, brown sugar, sucrose, glycerol, starch, corn starch, lactose,dextrin, molasses, and like carbohydrate sources. Preferred nitrogensources include 00 com steep liquor, yeast, autolyzed brewers yeast withmilk solids, soybean meal. cottonseed meal, corn meal, milk solids,pancreatic digest of casein, distillers solubles, animal peptoneliquors, meat and bonescraps, and like Combination of these carbon andnitrogen sources can be used advantageously. Trace metals, for example,zinc, magnesium, manganese, cobalt, iron, and the like, need not beadded to the fermentation media since tap water and unpurifiedingredients are used as media components.

Production of the compound of the invention can be efiected at anytemperature conductive to satisfactory growth of the microorganism, forexample, between about 18' and 40 C. and preferably between about 26 and30 C. Ordinary, optimum production of the compound is obtained in fromabout 2 to 10 days. The medium normally stays fairly close to neutral,or on the alkaline side, during the fermentation. The final pH isdependent, in part, on the initial pH of the culture medium which isadvantageously adjusted to about pH 68 prior to sterilization, and thebuffers present, if any.

When growth is carried out in large vessels and tanks, it is preferableto use the vegetative form of the microorganism for inoculation to avoida pronounced lag in the production of the new compound and the attendantinefficient utilization of the equipment. Accordingly, it is desirableto produce a vegetative inoculum in a nutrient broth culture byinoculating the broth culture with an aliquot from a soil or slantculture. When a young, active vegetative inoculum has thus been secured,it is transferred aseptically to large vessels or tanks. The medium inwhich the vegetative inoculum is produced can be the same as, orditferent'from, that utilized for the production of the new compound aslong as it is such that a good growth of the microorganism is obtained.

The new compound of the invention is a difunctional molecule having theempirical formula C l-N 0,. It has a basic function of about pKa'6.25-7.25 and an acidic function of about pKa' 9.009.60. It is highlysoluble in lower-alkanols, e.g., methanol, ethanol, isopropanol, thebutanols, and the like; lower-alkyl esters of lower alkanoic acids,e.g., ethyl acetate, n-butyl acetate, amyl acetate, and the like; andchlorinated loweralkanes, e.g., methylene chloride, chloroform, ethylenedichloride, and the like. It has some solubility in benzene and etherbut is insoluble in Skellysolve B (technical nhexane, B.P. 60-68 C.) andeyclohexane. is soluble at pHs less than 5.0 and greater than 9.5. Atthe isoelectric point (pH of ca. 8.3) its solubility in water is at aminimum.

In accordance with a preferred procedure for the recovery of the newcompound of the invention, the whole beer is adjusted, if necessary ordesirable, to a near neutral pH or below, suitably between pH 2 and pH4, heated to a temperature between 40 C. to 80' C. and filtered. Afilter aid, for example diatomite can be used. The filtered beer is thencooled to a conveniently obtained temperature, i.e. -5 C. The pH of thecooled filtrate is then adjusted to place the compound in thenon-protonated form. This is accomplished by neutralizing the solutionwith suitable base, for example, sodium hydroxide, to a pH greater thanpH 7.5, advantageously to a pH from 7.5 to 8.5. The resulting solutionis then extracted with a water-immiscible solvent and the new compoundrecovered from the solvent phase. If desired, the solvent phase can beacidified and the new compound recovered in the protonated form. Thiscan be accomplished by precipitating the new compound as an insolublesalt or by extracting the solvent extract with an aqueous solution of anacid which forms a water soluble salt, for example, hydrochloric acid,sulfuric acid, phosphoric acid, and acetic acid. Advantageously, thelatter is accomplished by adjusting the pH to less than 7.5, preferablyfrom pH 2 to pH 6. The salt is then recovered by evaporation.

If desired, the above-extraction procedure can be repeated as necessaryto effect desired purification before the salt is recovered. Also achange of water-immiscible solvent can be utilized to effect furtherpurification. For example, methylene chloride can be utilized to washout impurities from the aqueous solutions of the salt form or to extractthe free base from aqueous solutions ofthe nonprotonated compound.

The new compound of the invention can also be recovered from thefiltered beer by adsorption on cation In water it exchange resins. Boththe carboxylic and sulfonic acid types can be used. Suitable carboxylicacid resins include the polyacrylic acid resins obtained by thecopolymerization of acrylic acid and divinylbenzene by the proceduregiven on page 87 of Kunin, Ion Exchange Resins, 2nd Ed. (1958), JohnWiley and Sons, Inc. Carboxylic acid cation exchange resins of this typeare marketed under the trade names Amberlite IRC-50 and Zeokarb 226.Suitable sulfonic acid resins include nuclear sulfonated polystyreneresins cross-linked with divinylbenzene obtained by the procedure givenon page 84 of Kunin, supra. Sulfonated cation exchange resins of thistype are marketed under the trade names, Dowex-50, Amberlite IR-l20,Nalcite HCR, Chempro C-20, Permutit Q, and Zeokarb 225.

The protonated antibiotic is eluted from the resin with water at an acidpH, advantageously at a pH lower than the pKa' of the cation exchangeresin used. Satisfactory results are obtained with a pH of about 1 to 6.The excess acid in the eluate is neutralized to about pH 7.5 to 8.5 witha base, e.g., sodium hydroxide or a strongly basic anion exchange resin,and the antibiotic is extracted with a water-miscible solvent accordingto the process described above. Suitable anion exchange resins for thispurpose are obtained by chloromethylating by the procedure given onjages 88 and 97 of Kunin, supra, polystyrene crosslinked, if desired,with divinylbenzene prepared by the procedure given on page 84 of Kunin,supra, and quaternizing with trimethylamine, or dimethylethanolamine bythe procedure given on page 97 of Kunin, supra. Anion exchange resins ofthis type are marketed under the trade names Dowex 2, Dowex 20,Amberlite IRA-400, Duolite A-102, and Permutit 8-1.

The novel compound of the invention can also be recovered from harvestbeers and other aqueous solutions by adsorption on a surface activeadsorbent, for example, silicates, decolorizing carbon, or decolorizingresins, and eluting the adsorbed material with a solvent. Any of thesolvents mentioned above can be used. A suitable decolorizing resin isPermutit DR (US. Patent 2,702,263).

. The new compound of the invention can be purified by successivetransfers from protonated to nonprotonated forms and vice versa,especially with intervening other types of treatments, as for example,solvent extractions and washings, chromatography, and fractionalliquid-liquid extractions.

Fractional liquid-liquid extraction is accomplished in partitionchromatographic columns or in countercurrent distribution apparatus,using such solvent systems as Eethyl acetate-water 1:1 Cyclohexane,ethyl acetate, water l:l:l.5

The salts can be converted to the free base by neutralizing with analkali or by contacting with an anionic resin, advantageously to aboutpH 7.5 to 8.5. Specific acid salts can then be made by neutralizing thefree base with the appropriate acid to below about pH 7.5, andadvantageous- -ly to about pH 2 to pH 6. Suitable acids for this purposeinclude hydrochloric, sulfuric, phosphoric, acetic,

succinic, citric, oxalic, lactic, maleic, and fumaric, methanesulfonic,benzenesulfonic, hclianthic, Reineckefs azobenzcnesulfonic, picric, andlike acids.

The new compound of the invention, pactacin, has a broad antibacterialspectrum, and also inhibits the growth of fungi as shown in Tables TVand V.

TABLE IV.-ANTIBAGTERIAL ACTIVITY OF PACT ACIN MiC=Minimurn inhibitoryconcentration.

Activity determined in Difco brain heart infusion broth. according tothe method of Smith et 11., Antibiotics and Chemotherapy, volume 6,pages 135-142, 1956.

TABLE V.-ANTIFUNGAL ACTIVITY OF PACTACIN inhibition oi growth I Fungi1000 /ml. 10 /ini.

Noeordia asteroids: Blaetomycu dermnllildil Cocci ilohlu immilisGeolrielium sp llormodemirum campactum- J'Malophora urrucom Oryplococcmnee/ennui". Ilisioplasmo copeulatum. Sporotrichum sciienckil..lilonuporium opiospermum Triehoplyton rubrum Illierospomm canll TrieltoAyttm lnlerdigitale Candi a olbieans Abbott..- Trtchophvton rioloceumiililllllliiill 1 Complete inhibition. No inhibition.

The test compound is incorporated in agar in Petri dishes atconcentrations of 1000 and 10 mcg./ml. Suspensions of the test fungi arestreaked on the agar surface. After incubation for 72 hours at 28 C.,the Petri dishes are examined and the degree of inhibition of growthobserved.

The new compound of the invention is active against Bacillus subtilisand can be used for treating breeding places of silk worms to prevent orminimize infections caused by this organism. It can also be used tominimize or prevent odor in fish and fish crates caused by thisorganism. The new compound can be used as a disinfectant on variousdental and medical equipment contaminated with Staphylococcus albus orStaphylococcus aurcus; it can also be used as a disinfectant on washedand stacked food utensils contaminated with Staphylococcu: aureus.

The following examples are illustrative of the process and products ofthe present invention, but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example I (A) Fermentatlon.-A soil stock of Streptomyces pactum var.pactum, NRRL 2939, was ussecl to inoculate ten SOD-ml. Erlenmeyer flaskseach containing 100 m1. of seed medium consisting of the followingingredients:

Glucose monohydrate "grams-.. 25 Pharmamedia 1 "do-.. 25 Tap water, q.s"liters" 1 *Pharmamedia is an lndustrial grade of cottonseed flourproduced by Traders Oil Mill (20., FortWor-th, Texas.

The seed medium presterilization pH was 7.2. The seed was grown forthree days at 28' C. on a Gump rotary shaker operating a: 250 rpm.

'Dwoshskcflasksofthemdescribed above (IOOmL) were used to inoculate twoZO-litcr seed tanks containing 15 liters each of the following sterileseed medium:

Glucose monohydrate grams/liter 1O Corn steep liquor do 10 Pharmamediado 2 Wilson's Peptone Liquor No. 159 1 ..do 10 Lard oil ml./liter 2 Tapwater Balance Wilson's Peptone Liquor No. 159 is a preparation of ensymatieaily hydrolyzed proteins from animal origin.

The presteriiization pH of the seed tank medium was 7.2. Each seed tankwas grown for 24 hours at a tem perature of 28' C., aeration rate of 10standard liters/ min., and agitated at a rate of 400 rpm.

The two seed tanks, described above, were then used to inoculate two380liter fermentors containing 250 liters of the following sterilemedium:

Yeast (Pabst) grams/liter 25 Black strap molasses do 50 Glucosemonohydrate do 15 pH was adjusted to 6.4 with 50% aqueous NaOH thencalcium carbonate was added grams 5 Lard oil ml./liter.... 5 Tap waterBalance Each of the two fermentors was then grown for 5 days at atemperature of 32 C., aeration rate of standard liters/min, and agitatedat a rate of 280 r.p.m.

The biological assay and pH pattern of the above described fermentationswere as follows:

FIRST TANK B. lubttlls 1 pH hiounlts/nil.

SECOND TANK B. subti is Tissue eul- Tiours pill hiounits/mi. turo Iassay,

Kl! uJml.

70 e. a i 140 1. o 94 7. 6 18. 0 118 8. 2 230 40. 0

That dilution 0! 1 unit of material which eilects a 50% inhibition oiprotein synthesis KB Ulunitoimaterial 1000 (mg. or mi.)

(B) Extraction-Whole beer from the first tank (250 liters) was heated to60' C., adjusted to pH 2.55 with concentrated sulfuric acid, andfiltered using 4% filter aid. The clear beer (240 liters) was cooled,adjusted to pH 8.5 with 50% aqueous sodium hydroxide, and extracted oncewith 56 liters of ethyl acetate. The spent beer (240 liters) wasdiscarded. The ethyl acetate extract was washed with 6 liters of water;the wash was discarded. The washed ethyl acetate solution was thenbackextracted with water acidified to pH 1.95 with hydrochloric acid.The aqueous extract (16 liters) was adjusted to pH 5.5 with 10% aqueoussodium hydroxide and then freeze-dried to yield 41 grams (preparationDEG-71.1).

The second tank was processed in the same way to yield 22.7 grams(preparation DEG-766A).

The tissue culture assay on the two preparations, dc-' scribed above,was as follows:

Preparation: KBU/mg. DEG-71.1 29 DEG-76.6.. 91

(C) Purification.-These two preparations (DEG-7l.i+DEG-76.6A)

were pooled and subjected to further-fractionation by countcrcurrentdistribution using a solvent system of ethyl acetate and water in theratio of 1:1. Solids determina lions were run after 150 transfers andtubes 131 to 140, inclusive, were then pooled to give 8.4 grams(preparation AD-77.10) having a tis ue culture assay 200 KBU/mg. Sixgrams of this preparation was dissolved in a mixture of 15 ml. methylenechloride and 15 ml. acetone and pressed thru a Fiorisil (a mixture ofmagnesium and sodium trisilicates) column prepared by slurrying 300grams of Florisil in Skeliysolve B and pouring into a three inch (insidediameter) glass column; the column height was about eighteen inches. Thecolumn was eluted as shown in the following table:

FLO RISIL CHROMATOGRAPHY OF AD-77.10

Weight Tissue Fraction No. Eiuting solvent in mg. culture,

KB Uimg.

Skefiysolve Ii(88B) 8S li-Aoctone, 90: 10. BE-ll-Acetono, 80:10.

Still-Acetone, 80:3).

83il-Acetone, 75:25".-. 330 91 BSD-Acetone, 75:25 680 83 He'll-Acetone,10 30 240 91 SSH-Acetone, 7e: 60 143 SSH-Acetone, 65:35 20 BBB-Acetone,66:36. 10

Preparation AD-85-.6 from the above described Fiorisil column had anequivalent weight of 279 (indicating a molecular weight of about 558)and characteristic ultraviolet absorption spectrum in EtOH:

239.5 mp, a=51.22 264(sh.) mp, a=l4.81 313 mp, a=4.88 356 mp, a=3.25

0.01 ethanolic H 50 238 mp, a==50.45 262(sh.) mp, a =14.65 314 mp, ti-4.75 352 mp, a-3.5i

0.01 cthanollc KOH:

238.5 m a=56.38 264 mp, (1:16.01 322 mp, ar 6.05

a basic function of pKa' 7.25 in water and an acid function of pKa' 9.35in water, gave a negative ferric chloride test, and had a characteristicinfrared absorption spectrum (FIGURE 1 of the accompanying drawing) atthe following wave lengths expressed in reciprocal centimeters.

3333 M 1455 S (oil) 975 M 3175 -M- 1370 S (oil) 950 W 2890 S (oil) 1320S 940 W 2815 M (oil) 1285 S 920 W 1718 W 1247 S 870 W 1658 S --1205 S803 M 1592 S 1160 M 778 M 1575 S 1100 S 719 W (oil) 1515 S 1080 M 699 M1504 S 1038 M 685 M Frequency tolerances are 20 cm. in the 8800-2000 cm:range, cm: in the 000-1700 canrange, and 1-5 cm. in the 1700-700 cm.interval. The a ncing be tween ndiacent bands shall be as indicated inthe ahniation with n to erance of onofitth at the frequency tolerance.

Band intensities are indicated as "S," 1111' and W respectively, and areup roximnted in terms of the bachgrounds in the vicinity o the bands. An8" band is oi. the same order of intensity as the strongest band in thespectrum; "M" hands are between one-third and two-thirds as intense asthe strongest hand,- and W" bands are less than one-third as intense asthe strongest hand. These estimates are made on the basis of a percenttransmission scale.

Example II (A) Fermentarlom-Two 250-liter fermentations were preparedand run as described above in Example I. The assays on these tanks wereas follows:

(B) Ertrnc1ion.The whole beers from tanks RFD-9 and 11 were adjusted topH 2.7-2.8 with concentrated sulfuric.acid, heated to 60' C., pooled andcooled to 25' C. The pooled beer (500 liters) was then filtered using 4%filter aid and washed. The clear beer (510 liters) was adjusted to pH8.5 and extracted once with one-third volume. of ethyl acetate. Theresulting emuleion'was separated by centrifugation to yield 54 liters ofethyl acetate extract. Theethyl acetate was then back-extracted with 2.2liters of water acidified to pH 1.9 with hydrochioricacid. The'2.2liters of aqueous extract was adjusted to pH 5.5 with 50% aqueous sodiumhydroxide andfreeae-driedto yield 15 grams (preparation WTP-108-7).

The material balance is given in the following table:

grams) was extracted twice with 300 m1. portions 0! ethyl acetate. Theextracts (600 ml.) were pooled and concentrated in vacuo to a volume of50 ml. This solution was then purifiedover a Fiorisil column containing300 grams of'Fiorisil'which was siurried in Slt'cllysolve B. The columnwas eluted with 400 m1. portions of each of the following combinations:

Fractions 6 to 10, inclusive, were pooled and distributed through 400transfers in the Craig Countercurrent distribution apparatus using asystem of cyclohexane, ethyl acetate, and water in the ratios ofl:1:1.5. Fractions|33l to 370, inclusive, were combined and concentratedin vacuo to an aqueous solution which was lyophilized to yield 1.7 grams(preparation AD-107.1) assaying 3600:4000 B. subtilis biounits/mg., amelting point at 82-83 C., a characteristic ultra-violet absorptionspectrum in EtOH:

239.5 m a=52.65 264 (sh.) m :14.76 313 my, 0:521 356 my, a=3.38 0.01.ethanolic H 50 238 mu, a=49.42 262 mp, a: 14.61 314 my, a=4.40 352 my,a==3.45 0.01 ethanolie KOH:

- 264 III/1., a=l6.11 322 m a=6.05

an infrared absorption spectrum the same as preparation AD-85.6(described above in Example I, and as shown in FIGURE 1 of theaccompanying drawing), and the following elemental analysis:

Calculated for 0,11 ,140,: C, 59.98; H, 7.20; N, 10.00; 0, 22.83. Found:C, 60.33; H, 7.07; N, 9.34; O, 22.99.

Example III (A) Fermentation.-A fermentation, as described above inExample I, part A, was scaled up to a 5000 liter fermentor.

(B) Extractionr-The whole beer (4400 liters assaying 120 B. subtilirunits/ml.) was adjusted to pH 2.8 with concentrated sulfuric acid,heated to 60 C., cooled to 30 C., and filtered using 4% filter aid andwashed. The clear beer (4800 liters) was cooled to 5 C., ad justed to pH8.5 with 50% aqueous sodium hydroxide, and then extracted three timeswith ethyl acetate. The first ethyl acetate extract (450 liters) waskept separate. The second and third ethyl acetate extracts were pooled(3160 liters). The first extract was washed with water, adjusted to pH8.5 with 50% aqueous sodium hydroxide, and then back-extracted with a 2%volume of deionized water acidified to pH 1.7 with concentrated sulfuricacid. This aqueous extract (11 liters) was adjusted to pH 8.5 andback-extracted twice with ethyl acetate. These two ethyl acetateextracts were then pooled to give 8 liters of extract preparation (DMW-46.8).

10 The pooled second and third ethyl acetate extracts (3160 liters),described above, were concentrated to one half volume (1580 liters) andextracted with aqueous acid at pH 1.7 as described above for the firstethyl acetate extract. The resulting first aqueous extract was thenneutralized with 50% aqueous sodium hydroxide and freeze-dried(preparation DMW-46.11). The second aqueous extract was back-extractedwith ethyl acetate at pH 8.5. The ethyl acetate extract was dried byazeotropic distillation and evaporated to dryness (preparationDMW46.l3). Preparations DMW-46.1l, and DMW46.13 were then dissolved intoethyl acetate and the combined ethyl acetate solubles (BMW-46.17) wereused as the starting material for a three inch Florisil column whichcontained 1810 g. of Florisil and had a bed depth of thirty-two inchesand a liquid holdup of 3300 ml. The feed, a concentrate of preparationsDMW- 46.8 and BMW-46.17 (2.6 liters), was washed into the bed with oneliquid holdup volume of Skellysolve B (SSB) after first washing thecolumn with SSB. The column was eluted as follows:

One holdup volume of SSE-acetone (:10) One holdup volume of SSE-acetone(80:20) One holdup volume of SSB-acetone (75:25) Four holdup volumes ofSSE-acetone (70:30)

The starting rate of the efiluent off the column was 300 ml./min. butthe rate gradually dropped to ml./rnin. during the final stages of theelution. The acitve fractions were pooled to give 19 grams of solids(prep. DMW-53.2) assaying 2500 B. subtilis biounits/mg.

Preparation DMW-53.2 (19 grams), described above, was dissolved in 1liter of ether. The ether solution was dried with magnesium sulfate andevaporated to dryness. The slightly yellow material obtained was driedin vacuo at room temperature for two hours, yielding 12.2 grams(preparation AD139.2). Further purification of this preparation was byuse of a Florisil column prepared by slurrying 300 grams of Florisil inSkellysolve B and pouring the slurry into a 3'' LD. glass column. Thecolumn had a liquid holdup volume of 200 ml. Preparation AD-139.2 wasthen slurried in 20 ml. of methylene chloride and placed on the top ofthe column. The column was eluted with 200 ml. portions of the followingsolvent combinations:

SSH-(Acetone (70:30). SS 8+ Acetone (50:50)-

SSl3+Acetont (50:50). Acetone Acetone The most active fractions, 6, 7,8, and 9, assayed from 3400 to 4600 B. subtilis biounits/mg. Fraction 8was standardized at 3.4 B. subtilis biounits/mcg. Fraction 7 had anultraviolet absorption spectrum in ethanol 239 m 11:48.2 313-111 1,'a=4.6 355 m a=3.02

Total solids in mg.

1 1 Example IV A saturated solution of citric acid in ether was addedslowly to 30 ml. of an ether solution containing 400 mg. of preparationFraction 9 of Example III, Part C until there was no further formationof precipitate. The precipitate, a white amorphous material, wasfiltered off,

' washed with ether, and dried; yield 450 mg. About 300 Example V Asaturated solution of oxalic acid in ether was added to 25 ml. of ethercontaining 300 mg. of preparation Fraction 8 of Example III, Part C,until there was no further formation of precipitate and allowed to standat room temperature for ten minutes. The resultant precipitate wasfiltered off, washed wtih ether, and dried. The dried material (480 mg.)was reprecipitated from a methanol ether solution to give 220 mg. of awhite amorphous powder, preparationAD-3.1, which assayed 177 KBU/mg. intissue culture and 1250 mcg./mg. (free base) against the B. subtilis'bio assay, and had the following elemental analysis:

Calculated for C H N,0,-C,0 H C, 55.37; H, 6.51; N, 8.61; O, 29.51.Found: C, 54.85; H, 6.50; N, 8.73; O, 29.92 (by difierence).

Example Vl Hydrogen chloride was passed through 25 ml. of ether solutioncontaining 300 mg. of preparation Fraction 8 of Example III, Part C,until the formation of the white precipitate ceased. The precipitate wasfiltered otf, washed with ether, and dried to give 325 mg. of practacinhydrochloride, preparation AD-5.l, which assayed 43.5 KBU/mg. in tissueculture and 1175 meg/mg. (free base) against the B. subtilis bioassay,and had the following elemental analysis:

Calculated for C H N O -HCI: C, 56.32; H, 6.92; N, 9.39; 0, 21.44; Cl,5.94. Found: C, 56.18; H, 6.93; N, 8.75; Cl, 6.17; O, 21.97 (bydifference).

We claim:

1. A composition of matter assaying at least 20 meg/mg. of pactacin, acompound which (a) is effective in inhibiting the growth ofgram-positive and gram-negative bacteria, and

(b) is effective in inhibiting the growth of KB cells in tissue culture;and in its essentially pure cyrstalline form (c) is an amphotericsubstance with a basic function of pKa 6.25-7.25 and an acidic functionof pKa' 9.09.60;

(d) is highly soluble in lower-alkanols, lower-alkyl esters of loweralkanoic acids, and chlorinated loweralkanes,

(e) has a calculated empirical formula C H N O (f) has a molecularweight of about 558;

(g) has a characteristic ultra-violet absorption maxima as follows EtOH:

239.5 mg, a='-'1.22 264 (5b.) ma, a: 14.81 313 mn, a=4.88 356 m a=3.25

0.01 ethanolic H 50 238 m a='50.45 262 (5b.) my, 11:14.65 314 m (1:4.75352 mn, 0:3.51

0.01 ethanolic KOH:

238.5 mg, (1:56.33 264 m (1:16.01 322 my, a 6.05

and

(h) has a characteristic infrared absorption spectrum as shown in FIGURE1 of the accompanying drawing.

2. A compound, pactacin, according to claim 1 in its essentially purecrystalline form.

3. The citrate of pactacin as defined in claim 1.

4. The oxalate of pactacin as defined in claim 1.

5. The hydrochloride of pactacin as defined in claim 1.

6. A compound selected from the group consisting of pactacin accordingto claim 1 and the acid addition salts thereof.

7. A process which comprises cultivating Streptomyces pactum var. pactumin an aqueous nutrient medium under aerobic conditions until substantialactivity is imparted to said medium by production of pactacin.

8. A process which comprises cultivating Streptomyces pactum var. pactumin an aqueous nutrient medium containing a source of assimilablecarbohydrate and assimilable nitrogen under aerobic conditions untilsubstantial activity is imparted to said medium by production ofpactacin and isolating the pactacin so produced.

9. A process according to claim 8 in which the isolation comprisesextracting the medium with a water-immiscible solvent for pactacin andrecovering pactacin from the solvent extract.

10. A process according to claim 9, in which the recovery of thepactacin from the solvent extract is accomplished by fractionalliquid-liquid extraction.

11. A compound as defined in claim 1, pawtacin, in its essentially pureform.

References Cited by the Examiner Antimicrobial Agents and Chemotherapy,1961, pp. 191-204.

JULIAN S. LEVI'IT, Primary Examiner.

M. O. WOLK, L. GO'ITS, Examiners.

1. A COMPOSITION OF MATTER ASSAYING AT LEAST 20 MCG./MG. OF PACTACIN, ACOMPOUND WHICH (A) IS EFFECTIVE IN INHIBITING THE GROWTH OFGRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA, AND (B) IS EFFECTIVE ININHIBITING THE GROWTH OF KB CELLS IN TISSUE CULTURE; AND IN ITSESSENTIALLY PURE CRYSTALLINE FORM (C) IS AN AMPHOTERIC SUBSTANCE WITH ABASIC FUNCTION OF PKA'' 6.25-7.25 AND AN ACIDIC FUNCTION OF PKA''9.0-9.60; (D) IS HIGHLY SOLUBLE IN LOWER-ALKANOLS, LOWER-ALKYL ESTERS OFLOWER ALKANOIC ACIDS, AND CHLORINATED LOWERALKANES, (E) HAS A CALCULATEDEMPIRICAL FORMULA C28H40N4O8; (F) HAS A MOLDECULAR WEIGHT OF ABOUT 558;(G) HAS A CHARACTERISTIC ULTRA-VIOLET ABSORPTION MAXIMA AS FOLLOWS ETOH:239.5 M$,A=51.22 264 (SH.) M$,A=14.81 313 M$,A=4.88 356 M$,A=3.25 0.01ETHANOLIC H2SO4: 238 M$, A=50.45 262 (SH.) M$,A=14.65 314 M$,A=4.75 352M$,A=3.51 0.01 ETHANOLIC KOH: 238.5 M$,A=56.38 264 M$,A=16.01 322M$,A=6.05 AND (H) HAS A CHARACTERISTIC INFRARED ABSORPTION SPECTRUM ASSHOWN IN FIGURE 1 OF THE ACCOMPANYING DRAWING.